The isolation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological method for the production thereof, for example in the preparation of protein or peptide-based pharmaceutical compounds. Similarly there is also a need to separate biomolecules for analytical purposes in order to be able to quantify and identify the proteins and/or peptides present in a sample.
For identification and characterization of separated proteins mass spectrometry (MS) methods are normally used. A sample applied to a MALDI (Matrix Assisted Laser Desorption Ionization) MS target is only allowed to contain a limited number of peptides and similarly ESI (electrospray ionization) -MS can only accept a limited number of peptides per time unit. The sample is normally a very complex mixture containing many thousand of proteins which after digestion could easily correspond to one hundred thousand to more than one million peptides. There is therefore a need for rigorous separation of the peptides prior to MS characterization and quantification. A variety of different separation methods including electrophoretic and chromatographic methods can be used; normally multiple separation steps are required.
Generally electrophoretic techniques like isoelectric focusing (IEF) and sodium dodecyl sulphate (SDS) electrophoresis give, when used at the protein level in gel, much better resolution and protein yields than chromatographic alternatives. 2-D electrophoresis based on the combination of these two techniques, IEF and SDS, is also a commonly used approach when separation of very complex samples is conducted at the protein level.
In isoelectric focusing (IEF), the separation takes place in a pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. While other alternatives also exist, the pH gradients required in isoelectric focusing are in practice generated in two different ways: with the aid of a solution of carrier ampholytes or with an immobilized pH gradient. Carrier ampholytes are a spectrum of low molecular weight ampholytes with closely spaced isoelectric points. The carrier ampholytes give a good and regular conductivity at the isoelectric point and can carry the current during focusing.
In the case of an immobilized pH gradient (IPG) the charged or chargeable groups generating the pH gradient are bound either to the wall of a capillary system or to the matrix when some kind of gel is used to get convection stabilization. The immobilized charged or chargeable groups used are normally a limited number of carboxylic groups or amino groups with different pK-values distributed within or close to the pH gradient, which is to be generated. The concentration of the charged or chargeable groups is varied along the separation distance in a manner causing the pH at which the wall or the gel matrix has a zero net charge to increase from the anode to the cathode. A commercially available example of a system for generation of immobilized pH gradients is the IMMOBILINE II SYSTEM™(Amersham Biosciences, Uppsala, Sweden), wherein a pH gradient covalently attached to a polyacrylamide gel is formed. Immobilized pH gradients are truly stationary and today they are normally used together with carrier ampholytes. In this combination the immobilized gradient determines the resulting pH gradient, while the carrier ampholytes contribute with conductivity.
Focusing of peptides in presence of carrier ampholytes (PHARMALYTE™, AMPHOLINE™ or IPG (immobilized pH gradient)-buffer) result in sharper better focused peptide bands than when focusing is done in the absence of carrier ampholytes. The carrier ampholytes and the peptides both represents mixtures of large numbers of amphoteric compounds and the properties are partly very similar, which make it difficult to completely separate the to types of amphoteric compounds after finished focusing. A presently used approach is to trap the peptides on a reversed phase column and wash away the carrier ampholyte. However, this procedure results in the loss of some peptides together with the carrier ampholytes, while other carrier ampholyte compounds are bound with the peptides to the column. When released together with the peptides these latter ampholytes will generate a background in the mass spectrum used for quantification and/or identification of peptides.
Of existing methods for peptide separation, isoelectric focusing in IPG strips is the method, which gives the highest resolution and the best reproducibility. But as appears above, there is a need for improvement within this field.